寄生"螃蟹脚"的抗菌活性物质及其作用机理研究

Viscum articulatum Burm.f. is a parasitic on the teas, fruit trees, Chinese sweet gum and other trees. However, only Viscum articulatum Burm.f. parasited on the teas has a strong antibacterial action, and which has been confirmed by practice. Antimicrobial constituents and their mechanism of Viscum articulatum Burm.f., so far, there have been no reported at home and abroad. Based on the preliminary study, this project is launched by using different polar solvent and column integrated technology and in conjunction with high-throughput screening technology to track the antibacterial constituents. The composition and structure of the antimicrobial substances are clearly defined by using HPLC-MS, NMR, IR, UV and in combination with their physicochemical properties, the special mechanism of antibacterial constituents will elucidate from the molecular and cellular levels such as cell morphology, cell tissue, nucleic acid, protein by using atomic force microscopy, transmission electron microscopy, conductivity meter, enzymology and molecular biology. This project will prove the antimicrobial substances of Viscum articulatum burm f., reveal the molecular mechanism of antibacterial constituents and interprete the molecular structure of the antibacterial constituents and their structure-activity relationship, and which will provide the theoretical basis and scientific foundation to exploit and utilize the Viscum articulatum Burm.f. resource in Yunnan deeply, as well as provide a new clue and theoretical foundation for screening and mechanism of other natural antimicrobial constituents. Therefore, studying the antibacterial activity and its mechanism of action of the parasitic Viscum articulatum burm f. is highly valuable for academic study and prospects of application.

螃蟹脚是一种寄生于茶树、果树、枫香树等树上的寄生植物，但唯有寄生在茶树上的"螃蟹脚"才具有高效抗抑菌作用且已被实践所证实，但对其抗菌物质及其作用机理的研究迄今尚无报道。本项目在前期研究基础上，采用极性不同溶剂及色谱柱集成技术对"螃蟹脚"逐级分离，同时配合高通量筛选技术追踪活性抗菌成分。运用HPLC-MS、NMR、IR及UV等波谱技术并结合理化分析鉴定抗菌单体化合物，明确抗菌物质组成和结构。采用原子力显微镜、透射电镜、电导仪、酶学及分子生物学等技术从供试菌形态，组织，核酸、蛋白质等分子和细胞水平阐明其特殊抗菌作用机理。通过研究探明"螃蟹脚"抗菌的物质基础，揭示螃蟹脚抗菌的作用机理，阐释"螃蟹脚"抗菌活性成分的分子结构及其构效关系，为"螃蟹脚"资源的深度开发和利用提供理论基础和科学依据，同时为其他天然抑菌活性成分的筛选及机理研究提供新的思路和理论依据，研究成果具有重要的学术价值和应用前景。

广谱、高效、低毒、安全的抗菌防腐物质是世界各国研究的热点。本项目以茶寄生“螃蟹脚”为研究对象，运用高通量活性追踪方法，系统评价“螃蟹脚”不同提取物体外抑菌水平，利用现代天然产物分离和结构鉴定手段，对“螃蟹脚”的活性抗菌物质结构和作用机理进行系统研究。取得的主要研究结果：1.“螃蟹脚”95%乙醇、75%乙醇以及蒸馏水提取物对金黄色葡萄球菌、大肠杆菌、李斯特杆菌、铜绿假单胞菌、鼠伤寒沙门氏菌、纳豆芽孢杆菌以及变异微球菌均有明显抑制作用。在相同浓度下，95%乙醇扁枝槲寄生提取物抑菌效果＞75%乙醇扁枝槲寄生提取物抑菌效果＞蒸馏水扁枝槲寄生提取物抑菌效果。2.“螃蟹脚”提取物能维持冷鲜肉糜感官性状稳定，减少水分散失和汁液流出，延缓pH值和TVB-N数值的上升(P＜0.05)，抑制肉糜中的菌落总数、肠杆菌及假单胞菌的数量的增殖，将冷鲜猪肉糜的货架期延长4-6天；3.“螃蟹脚”乙酸乙酯提取物中主要为槲寄生新甙、柚皮素、圣草酚、高圣草素、圣草酚-7-O-β-D-葡萄糖甙等化合物,槲寄生新甙在“螃蟹脚”的抗菌活性中具有显著的作用。4.通过原子力显微镜、激光共聚焦显微镜、电镜观察、生化试验等探明槲寄生新甙通过多个作用点对供试菌生长产生影响，如作用于细胞壁、细胞膜、DNA等，从而造成菌体代谢紊乱、结构异常而解体死亡。本项目研究结果为开发新型天然保鲜剂奠定理论基础，为“螃蟹脚”的深度开发和利用提供科学依据。本项目培养研究生5人，已发表论文9篇，其中SCI论文3篇。